Light emitting microorganisms and cells for diagnosis and therapy of tumors

ABSTRACT

Provided are diagnostic and pharmaceutical compositions containing a microorganism or a cell containing a DNA molecule encoding a detectable protein or a protein that a detectable signal, such as a luminescent or fluorescent protein. Methods of tumor targeting and tumor imaging using the microorganisms and cells are provided. Also provided are therapeutic methods in which the microorganisms and cells, which can encoded a therapeutic protein, such as a cytotoxic or cytostatic protein, are administered.

This application is a continuation of U.S. application Ser. No.10/189,918, filed Jul. 3, 2002 now abandoned, to Aladar Szalay, entitled“Light emitting microorganisms and cells for diagnosis and therapy oftumors,” the subject matter of which is incorporated herein in itsentirety. Benefit of priority under 35 U.S.C. §120 is claimed.

The present invention relates to diagnostic and pharmaceuticalcompositions comprising a microorganism or cell containing a DNAsequence encoding a detectable protein or a protein capable of inducinga detectable signal, e.g. a luminescent or fluorescent protein. Thepresent invention also relates to the use of said microorganism or cellfor tumor-targeting or tumor-imaging. For therapeutic uses, saidmicroorganism or cell additionally contain an expressible DNA sequenceencoding a protein suitable for tumor therapy, e.g. a cytotoxic orcytostatic protein.

Presence of bacteria in tumors was reported approximately fifty yearsago. Several publications substantiated the earlier clinical findingsthat unexpectedly large numbers of bacteria were discovered in excisedtumors from human patients. Investigators argue that chronic infectionsmay predispose cells to malignant growth. Chronic infections of variousstrains of Chlamydia have been associated with lung and cervical canceras well as malignant lymphoma. Another well described associationbetween the presence of a specific bacterial species and cancerdevelopment is Helicobacter pylori in patients with gastric ulcers.Elevated levels of H. pylori-associated antibodies have been found inpatients with duodenal ulcer and gastric adenocarcinoma. Theseobservations demonstrate a concomitant presence of bacteria at tumorsites; however, it was yet not clear whether the microorganisms were thecause of tumor formation or whether the tumorous tissues were moresusceptible to bacterial colonization. Intravenously injected strictanaerobic bacteria, Clostridium pasteurianum, into mice replicatedselectively in the tumor suggesting a hypoxic microenvironment in thenecrotic center. Intravenous injection of attenuated Salmonellatyphimurium mutants resulted in elevated bacterial titers in the tumortissues in comparison to the other organs of mice upon histologic andbacteriologic analyses.

Similarly, the presence of virus particles was reported in excised humanbreast tumors as early as 1965. More recently, based on polymerase chainreaction (PCR) data, the human papillomavirus has been claimed to beassociated with anogenital tumors and esophageal cancers, breastcancers, and most commonly, cervical cancers. In addition, the presenceof hepatitis C virus in human hepatocellular carcinoma, Epstein-Barrvirus in squamous cell carcinoma in Kimura's disease, mouse mammarytumor virus-like particles (MMTV) in human breast cancer, SV40 virus inmacaque astrocytoma, and herpesvirus in turtle fibropapilloma has alsobeen reported. Surprisingly, the concentration of virus particles in thetumors shows variations among patients. The presence of humanpapillomavirus in squamous cell carcinomas of the esophagus ranges from0 to 72% (10-15). In contrast to tumor tissues, no virus particles havebeen found in tumor-free areas of the esophageal epithelium of the samepatient suggesting that the virus particles are located only in thetumor tissues.

However, so far it could not undoubtedly been shown whether the abovediscussed microorganisms are responsible for the development ofdisorders like tumors (except for papillomaviruses) or whether, e.g.,tumors can attract and/or protect viruses or bacteria. Accordingly,there was no basis for the use of such microorganisms for the diagnosisor therapy of tumors. Conventional tumor diagnostic methods, such as MRI(Magnetic Resonance Imaging) and therapeutic methods, e.g. surgery, areinvasive and not very sensitive.

Therefore, it is the object of the present invention to provide a meansfor the efficient and reliable diagnosis as well as the therapy oftumors which overcomes the disadvantages of the diagnostic andtherapeutic approaches presently used.

According to the present invention this is achieved by the subjectmatters defined in the claims. When Vaccinia virus (LIVP strain)carrying the light emitting fusion gene construct rVV-ruc-gfp wasinjected intravenously into nude mice, the virus particles were found tobe cleared from all internal organs within 4 days, as determined byextinction of light emission. In contrast, when the fate of the injectedVaccinia virus was similarly followed in nude mice bearing tumors grownfrom subcutaneously implanted C6 rat glioma cells, virus particles werefound to be retained over time in the tumor tissues, resulting inlasting light emission. The presence and amplification of thevirus-encoded fusion proteins in the same tumor were monitored in liveanimals by observing GFP fluorescence under a stereomicroscope and bycollecting luciferase-catalyzed light emission under a low-lightvideo-imaging camera. Tumor-specific light emission was detected 4 daysafter viral injection in nude mice carrying subcutaneous C6 gliomaimplants ranging in size from 25 to 2500 mm³. The signal became moreintense after the 4th postinjection day and lasted for 30 to 45 days,indicating continued viral replication. Tumor accumulation ofrVV-ruc-gfp virus particles was also seen in nude mice carryingsubcutaneous tumors developed from implanted PC-3 human prostate cells,and in mice with orthotopically implanted MCF-7 human breast tumors.Further, intracranial C6 rat glioma cell implants in immunocompetentrats and MB-49 human bladder tumor cell implants in C57 mice were alsotargeted by the Vaccinia virus. Cross sections of a C6 glioma revealedthat light emission was clustered in “patches” at the periphery of thetumor where the fast-dividing cells reside. In contrast, cross sectionsof breast tumors revealed that fluorescent “islands” were distributedthroughout the tumors. In addition to primary breast tumors, smallmetastatic tumors were also detected externally in the contralateralbreast region, as well as in nodules on the exposed lung surface,suggesting metastasis to the contralateral breast and lung. In summary,light-emitting cells or microorganims, e.g. Vaccinia virus can be usedto detect and treat primary and metastatic tumors.

Similar results were obtained with light-emitting bacteria (Salmonella,Vibrio, Listeria, E. coli) which were injected intravenously into miceand which could be visualized in whole animals under a low light imagerimmediately. No light emission was detected twenty-four hours afterbacterial injection in both athymic (nu/nu) mice and immunocompetent C57mice as a result of clearing by the immune system. In the cutaneouswound of an intravenously injected animal, the bacterial light emissionincreases and remains detectable up to six days post-injection. In nudemice bearing tumors developed from implanted C6 glioma cells, lightemission was abolished from the animal entirely twenty-four hours afterdelivery of bacteria, similar to mice without tumors. However,forty-eight hours post-injection, unexpectedly, a strong, rapidlyincreasing light emission originated only from the tumor regions wasobserved. This observation indicates a continuous bacterial replicationin the tumor tissue. The extent of light emission is dependent on thebacterial strain used. The homing-in process together with the sustainedlight emission was also demonstrated in nude mice carrying prostate,bladder, and breast tumors. In addition to primary tumors, metastatictumors could also be visualized as exemplified in the breast tumormodel. Tumor-specific light emission was also observed inimmunocompetent C57 mice with bladder tumors as well as in Lewis ratswith brain glioma implants. Once in the tumor, the light-emittingbacteria were not observed to be released into the circulation and tore-colonize subsequently implanted tumors in the same animal. Further,mammalian cells expressing the Ruc-GFP fusion protein, upon injectioninto the bloodstream, were also found to home into and propagate inglioma tumors.

These findings open the way for (a) designing multifunctional viralvectors useful for the detection of tumors based on signals like lightemission and/or for suppression of tumor development and/or angiogenesissignaled by, e.g., light extinction and (b) the development ofbacterium- and mammalian cell-based tumor targeting systems incombination with therapeutic gene constructs for the treatment ofcancer. These systems have the following advantages: (a) They target thetumor specifically without affecting normal tissue; (b) the expressionand secretion of the therapeutic gene constructs are, preferably, underthe control of an inducible promoter, enabling secretion to be switchedon or off; and (c) the location of the delivery system inside the tumorcan be verified by direct visualization before activating geneexpression and protein delivery.

Accordingly, the present invention relates to a diagnostic orpharmaceutical composition comprising a microorganism or cell containinga DNA sequence encoding a detectable protein or a protein capable ofinducing a detectable signal.

Any microorganism or cell is useful for the diagnostic method of thepresent invention, provided that they replicate in the organism, are notpathogenic for the organism e.g. attenuated and, are recognized by theimmune system of the organism, etc.

In a preferred embodiment, the diagnostic or pharmaceutical compositioncomprises a microorganism or cell containing a DNA sequence encoding aluminescent and/or fluorescent protein.

As used herein, the term “DNA sequence encoding a luminescent and/orfluorescent protein” also comprises a DNA sequence encoding aluminescent and fluorescent protein as fusion protein.

In an alternative preferred embodiment, the diagnostic or pharmaceuticalcomposition of the present invention comprises a microorganism or cellcontaining a DNA sequence encoding a protein capable of inducing asignal detectable by magnetic resonance imaging (MRI), e.g. metallbinding proteins. Furthermore, the protein can bind contrast agents,chromophores, ligands or compounds required for visualization oftissues.

Preferably, for transfecting the cells the DNA sequences encoding aluminescent and/or fluorescent protein are present in a vector or anexpression vector. A person skilled in the art is familiar with examplesthereof. The DNA sequences can also be contained in a recombinant viruscontaining appropriate expression cassettes. Suitable viruses that maybe used in the diagnostic or pharmaceutical composition of the presentinvention include baculovirus, vaccinia, sindbis virus, Sendai virus,adenovirus, an AAV virus or a parvovirus, such as MVM or H-1. The vectormay also be a retrovirus, such as MoMULV, MoMuLV, HaMuSV, MuMTV, RSV orGaLV. For expression in mammals, a suitable promoter is e.g. humancytomegalovirus “immediate early promoter” (pCMV). Furthermore, tissueand/or organ specific promoters are useful. Preferably, the DNAsequences encoding a luminescent and/or fluorescent protein areoperatively linked with a promoter allowing high expression. Suchpromoters, e.g. inducible promoters are well-known to the person skilledin the art.

For generating the above described DNA sequences and for constructingexpression vectors or viruses which contain said DNA sequences, it ispossible to use general methods known in the art. These methods includee.g. in vitro recombination techniques, synthetic methods and in vivorecombination methods as described in Sambrook et al., MolecularCloning, A Laboratory Manual, 2^(nd) edition (1989) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., for example. Methods oftransfecting cells, of phenotypically selecting transfectants and ofexpressing the DNA sequences by using the above described vectors areknown in the art.

The person skilled in the art knows DNA sequences encoding luminescentor fluorescent proteins that can be used in the diagnostic orpharmaceutical of the present invention. During the past decade, theidentification and isolation of structural genes encoding light-emittingproteins from bacterial luciferase from Vibrio harveyi (Belas et al.,Science 218 (1982), 791-793) and from Vibrio fischerii (Foran and Brown,Nucleic acids Res. 16 (1988), 177), firefly luciferase (de Wet et al.,Mol. Cell. Biol. 7 (1987), 725-737), aequorin from Aequorea Victoria(Prasher et al., Biochem. 26 (1987), 1326-1332), Renilla luciferase fromRenilla reniformis (Lorenz et al., PNAS USA 88 (1991), 4438-4442) andgreen fluorescent protein from Aequorea victoria (Prasher et al., Gene111 (1987), 229-233) have been described that allow the tracing ofbacteria or viruses based on light emission. Transformation andexpression of these genes in bacteria allows detection of bacterialcolonies with the aid of the low light imaging camera or individualbacteria under the fluorescent microscope (Engebrecht et al., Science227 (1985), 1345-1347; Legocki et al., PNAS 83 (1986), 9080-9084;Chalfie et al., Science 263 (1994), 802-805).

Luciferase genes have been expressed in a variety of organisms. Promoteractivation based on light emission, using lux AB fused to thenitrogenase promoter, was demonstrated in Rhizobia residing within thecytoplasm of cells of infected root nodules by low light imaging(Legocki et al., PNAS 83 (1986), 9080-9084; O'Kane et al., J. Plant Mol.Biol. 10 (1988), 387-399). Fusion of the lux A and lux B genes resultedin a fully functional luciferase protein (Escher et al., PNAS 86 (1989),6528-6532). This fusion gene (Fab2) was introduced into Bacillussubtilis and Bacillus megatherium under the xylose promoter and then fedinto insect larvae and was injected into the hemolymph of worms. Imagingof light emission was conducted using a low light video camera. Themovement and localization of pathogenic bacteria in transgenicarabidopsis plants, which carry the pathogen-activated PALpromoter-bacterial luciferase fusion gene construct, was demonstrated bylocalizing Pseudomonas or Erwinia spp. infection under the low lightimager as well as in tomato plant and stacks of potatoes (Giacomin andSzalay, Plant Sci. 116 (1996), 59-72).

All of the luciferases expressed in bacteria require exogenously addedsubstrates such as decanal or coelenterazine for light emission. Incontrast, while visualization of GFP fluorescence does not require asubstrate, an excitation light source is needed. More recently, the genecluster encoding the bacterial luciferase and the proteins for providingdecanal within the cell, which includes luxCDABE was isolated fromXenorhabdus luminescens (Meighen and Szittner, J. Bacteriol. 174 (1992),5371-5381) and Photobacterium leiognathi (Lee et al., Eur. J. Biochem.201 (1991), 161-167) and transferred into bacteria resulting incontinuous light emission independent of exogenously added substrate(Fernandez-Pinas and Wolk, Gene 150 (1994), 169-174). Bacteriacontaining the complete lux operon sequence, when injectedintraperitoneally, intramuscularly, or intravenously, allowed thevisualization and localization of bacteria in live mice indicating thatthe luciferase light emission can penetrate the tissues and can bedetected externally (Contag et al., Mol. Microbiol. 18 (1995), 593-603).

Preferably, the microorganism is a bacterium, e.g. attenuated.Particularly preferred is attenuated Salmonella thyphimurium, attenuatedVibrio cholerae or attenuated Listeria monocytogenes or E. coli.Alternatively, viruses such as Vaccinia virus, AAV, a retrovirus etc.are also useful for the diagnostic and therapeutic compositions of thepresent invention. Preferably, the virus is Vaccinia virus.

Preferably, the cell of the diagnostic or therapeutic composition of thepresent invention is a mammalian cell such as stem cells which can beautologous or heterologous concerning the organism.

In a further preferred embodiment of the diagnostic or therapeuticcomposition of the present invention the luminescent or fluorescentprotein is a luciferase, green fluorescent protein (GFP) or redfluorescent protein (RFP).

In a particularly preferred embodiment, the microorganism or cell of thediagnostic or pharmaceutical composition of the present inventionadditionally contains a gene encoding a substrate for the luciferase. Inan even more preferred embodiment, the microorganism or cell of thediagnostic or pharmaceutical composition of the present inventioncontains a ruc-gfp expression cassette which contains the Renillaluciferase (ruc) and Aequorea gfp cDNA sequences under the control of astrong synthetic early/late (PE/L) promoter of Vaccinia or the luxCDABEcassette.

A preferred use of the microorganisms and cells described above is thepreparation of a diagnostic composition for tumor-imaging. Thediagnostic composition of the present invention can be used e.g. duringsurgery, to identify tumors and metastasis. Furthermore, the diagnosticcomposition of the present invention is useful for monitoring atherapeutic tumor treatment. Suitable devices for analyzing thelocalization or distribution of luminescent and/or fluorescent proteinsin an organism, organ or tissue are well known to the person skilled inthe art and, furthermore described in the literature cited above as wellas the Examples, below. Additionally, the microorganisms and cells canbe modified in such a way that they bind metals and consequently areuseful in MRI technology to make this more specific.

The present invention also relates to a pharmaceutical compositioncontaining a microorganism or cell as described above, wherein saidmicroorganism or cell furthermore contains one or more expressible DNAsequence(s) encoding (a) protein(s) suitable for tumor therapy and/orelimination of metastatic tumors, such as a cytotoxic protein, acytostatic protein, a protein inhibiting angiogenesis, or a proteinstimulating apoptosis. Such proteins are well-known to the personskilled in the art. Furthermore, the protein can be an enzyme convertingan inactive substance (pro-drug) administered to the organism into anactive substance, i.e. toxin, which is killing the tumor or metastasis.For example, the enzyme can be glucuronidase converting the less toxicform of the chemotherapeutic agent glucuronyldoxorubicin into a moretoxic form. Preferably, the gene encoding such an enzyme is directed bya promoter which is inducible additionally ensuring that the conversionof the pro-drug into the toxin only occurs in the target tissue, i.e.tumor. Such promoters are e.g. IPTG-, antibiotic-, heat-, pH-, light-,metal-, aerobic-, host cell-, drug-, cell cycle- or tissuespecific-inducible promoters. Additional examples of suitable proteinsare human endostatin and the chimeric PE37/TGF-alpha fusion protein.Endostatin is a carboxyterminal peptide of collagen XVIII which has beencharacterized (Ding et al., PNAS USA 95 (1998), 10443). It has beenshown that endostatin inhibits endothelial cell proliferation andmigration, induces GI arrest and apoptosis of endothelial cells invitro, and has antitumor effect in a variety of tumor models.Intravenous or intramuscular injection of viral DNA and cationicliposome-complexed plasmid DNA encoding endostatin result in limitedexpression levels of endostatin in tumors. However intratumoralinjection of purified endostatin shows remarkable inhibition of tumorgrowth. Pseudomonas exotoxin is a bacterial toxin secreted byPseudomonas aeruginosa. PE elicits its cytotoxic effect by inactivatingelongation factor 2 (EF-2), which results in blocking of proteinsynthesis in mammalian cells. Single chain PE is functionally dividedinto three domains: domain Ia is required for binding to cell surfacereceptor, domain II is required for translocating the toxin into thetarget cell cytosol, and domain III is responsible for cytotoxicity byinactivating EF-2. PE40 is derived from wild type Pseudomonas exotoxinthat lacks the binding domain Ia. Other proteins such as antibodyfragments or protein ligands can be inserted in place of the bindingdomain. This will render the PE40-ligand fusion protein specific to itsreceptor. One of the highly specific engineered chimeric toxins is theTGF alpha/PE40 fusion protein, where the C-terminus of TGF alphapolypeptide has been fused in frame with the N-terminus of the PE40protein. TGF alpha is one of the ligands of epidermal growth factorreceptor (EGFR), which has been shown to be preferentially expressed onthe surface of a variety of tumor cells. TGF alpha-PE40 fusion proteinhas been shown to be highly toxic to tumor cells with elevated EGFRs onthe cell surface and while it is less toxic to nearby cells displayingfewer numbers of surface EGFR. The toxicity of TGF alpha-PE40 chimericprotein is dependent on a proteolytic processing step to convert thechimeric protein into its active form, which is carried out by thetarget. To overcome the requirement for proteolysis, a new chimerictoxin protein that does not require processing has been constructed byTheuer and coworkers (J. Biol. Chem. 267 (1992), 16872). The novelfusion protein is termed PE37/TGF alpha, which exhibited higher toxicityto tumor cells than the TGF alpha-PE40 fusion protein.

Thus, in a preferred embodiment of the pharmaceutical composition, theprotein suitable for tumor therapy is endostatin (for inhibition oftumor growth) or recombinant chimeric toxin PE37/transforming growthfactor alpha (TGF-alpha) (for cytotoxicity to tumor cells).

Moreover, the delivery system of the present application even allows theapplication of compounds which could so far not be used for tumortherapy due to their high toxicity when systemically applied. Suchcompounds include proteins inhibiting elongation factors, proteinsbinding to ribosomal subunits, proteins modifying nucleotides,nucleases, proteases or cytokines (e.g. IL-2, IL-12 etc.), sinceexperimental data suggest that the local release of cytokines might havea positive effect on the immunosuppressive status of the tumor.

Furthermore, the microorganism or cell can contain a BAC (BacterialArtificial Chromosome) or MAC (Mammalian Artificial Chromosome) encodingseveral or all proteins of a specific pathway, e.g. anti-angionesis,apoptosis, woundhealing-pathway or anti-tumor growth. Additionally thecell can be cyber cell or cyber virus endocing these proteins.

For administration, the microorganisms or cells of the present inventionare preferably combined with suitable pharmaceutical carriers. Examplesof suitable pharmaceutical carriers are well known in the art andinclude phosphate buffered saline solutions, water, emulsions, such asoil/water emulsions, various types of wetting agents, sterile solutionsetc. Such carriers can be formulated by conventional methods and can beadministered to the subject at a suitable dose. Administration of themicroorganisms or cells may be effected by different ways, e.g. byintravenous, intraperetoneal, subcutaneous, intramuscular, topical orintradermal administration. The preferred route of administration isintravenous injection. The route of administration, of course, dependson the nature of the tumor and the kind of microorganisms or cellscontained in the pharmaceutical composition. The dosage regimen will bedetermined by the attending physician and other clinical factors. As iswell known in the medical arts, dosages for any one patient depends onmany factors, including the patient's size, body surface area, age, sex,the particular compound to be administered, time and route ofadministration, the kind, size and localization of the tumor, generalhealth and other drugs being administered concurrently.

Preferred tumors that can be treated with the microorganisms or cells ofthe present invention are bladder tumors, breast tumors, prostatetumors, glioma tumors, adenocarcinomas, ovarial carcinomas, andpancreatic carcinomas; liver tumors, skin tumors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: External Imaging of GFP Expression in Subcutaneous C6 GliomaTumors in Nude Mice

C6 glioma cells (5×10⁵) were implanted subcutaneously into the rightlateral thigh. At designated days after tumor cell implantation, theanimals were infected intravenously with 1×10⁸ pfu of rVV-ruc-gfp virusparticles. GFP expression was monitored under a fluorescencestereomicroscope. Bright field (top), fluorescence (middle), and brightfield, fluorescence overlay (bottom) images of subcutaneous glioma tumorare shown. GFP signal can be observed in tumors as small as 22 mm³ insize (B-B″), or as old as 18 days (about 2500 mm³ in size) (A-A″). Inolder tumors, GFP expression was seen in “patch”-like patterns(indicated by arrows in A′). Marker gene expression in the tumor of thesame animal can be monitored continuously 4 (C-C″), 7 (D-D″), and 14(E-E″) days after intravenous viral injection. (Bars=5 mm.)

FIG. 2: Visualization of Tumor Angiogenesis

C6 glioma cells (5×10⁵) were implanted subcutaneously into the rightlateral thigh of nude mice. Ten days after tumor cell implantation, theanimals were infected intravenously with 1×10⁸ pfu of rVV-ruc-gfp. GFPexpression was monitored 7 days post-viral injection. Vascularization atthe surface of the subcutaneous C6 glioma tumor is shown against thebright green fluorescent background in the tumor followingVaccinia-mediated gene expressions. Bright field (A), fluorescence (B),and bright field, fluorescence overlay (C) images of subcutaneous gliomatumor are illustrated. (Bars=5 mm.)

FIG. 3: Expression of GFP in Subcutaneous Glioma Tumor of the SameAnimal

Five days after the subcutaneous implantation of 5×10⁵ C6 glioma cellsinto the right lateral thigh, 10⁸ of rVV-ruc-gfp virus particles wereinjected intravenously. Five days after viral injection, the animal wasanesthetized and sacrificed for analysis of GFP expression underfluorescence microscope. The tumor was visualized externally (A-A″),with the overlying skin reflected (B-B″), in cross section (C-C″), andin the amputated leg (D-D″). Bright field (A), fluorescence (B), andbright field, fluorescence overlay (C) images of subcutaneous gliomatumor are illustrated. The strongest GFP expressions are seen as patcheslocated along the outer surface of the tumor on the right (double arrowsin C-C″). Sharp difference of GFP expression in tumor tissue and in thenormal muscle tissue (arrows in D-D″) is clearly visible. Asterisks markthe reflected skin (B-B″ and D-D″). (Bars=5 mm.)

FIG. 4: Bright Field (A) and Fluorescence (B) Images of Tumor CellsExpressing GFP

Frozen sections (30 μm thick) of the glioma tumor tissues were preparedfrom a nude mouse that has been intravenously injected with 1×10⁸ ofrVV-rucgfp virus particles. (Bars=50 μm.)

FIG. 5: Low Light Image of the Anesthetized Nude Mouse to Indicate theLocation of Renilla luciferase-Triggered Light Emission in the Presenceof Intravenously Injected Substrate Coelenterazine (5 μg EthanolSolution)

FIG. 6: Monitoring Tumor-Specific Viral Infection Based on GFP GeneExpression in a Variety of Tumor Models

GFP gene expression was monitored in a variety of tumor models includingsubcutaneous PC-3 human prostate tumor (A-A″) and MCF-7 human breasttumor (B-B″) in nude mice, intracranial C6 rat glioma tumor (C-C″,arrows indicate the location of the tumor) in Lewis rats, and MB-49human bladder tumor (D-D″) in C57 mice. Animals were monitored 7 daysafter intravenous injections of 1×10⁸ of rVV-ruc-gfp virus particles.Bright field (top), fluorescence (middle), and bright field,fluorescence overlay (bottom) images of the tumor are illustrated.(Bars=5 mm.)

FIG. 7: Monitoring Vaccinia-Mediated GFP Expression in a Breast TumorModel

Nude mouse carrying breast tumor was injected intravenously with 1×10⁸of rVV-ruc-gfp virus particles. Both the primary tumor (A-A″, B-B″, andC-C″) and the metastasized tumor (D-D″, E-E″, and F-F″) were visualizedexternally (A-A″ and D-D″), with overlying skin removed (B-B″ and E-E″),and when they were split open (C-C″ and F-F″) in a set of bright field,fluorescence (′) and bright field, fluorescence overlay (″) images. GFPexpression in lung metastases in the same animal was also visualized(G-G″). (Bars=5 mm (A-A″ to F-F″), and Bars=1 mm (G-G″).

FIG. 8: Visualization of the Clearance of Light Emitting Bacteria FromNude Mice Based on the Detection of Light Emission Under the Low LightImager

Nude mice were intravenously injected with 10⁷ cells of attenuated S.typhimurium (A, B) and V. cholera (C, D). Both strains were transformedwith pLITE201 carrying the lux operon. Photon collection was done 20 min(A, C) and 2 days (B, D) after bacterial injections.

FIG. 9: Homing of Glioma Tumors by Attenuated Bacteria

Nude mice with a C6 glioma tumor in the right hind leg wereintravenously injected with 10⁷ attenuated S. typhimurium (A-D) and withV. cholera (E-H) both transformed with pLITE201 plasmid DNA encoding thelux operon. Photon collection was carried out for one minute under thelow light imager. Mice injected with S. typhimurium exhibitedluminescence immediately through the whole animal (A). In contrast,luminescence in the mice injected with V. cholera was visible in theliver area (E). Two days after bacterial injection, both groups of micedemonstrated luminescence only in the tumor region (B, F). The lightemission in the tumors infected with S. typhimurium slowly diminishedfour (C) and six (D) days after bacterial injection. Tumors infectedwith V. cholera showed enormously increased light emission four (G) andsix (H) days after injection suggesting continued replication of thebacteria in the tumor tissues.

FIG. 10: Homing in of Bacteria onto Breast Tumors

Nude mice with breast tumors in the right breast pad were intravenouslyinjected with 10⁷ attenuated V. cholera (A-D) and with 10⁷ E. coli (E-F)transformed with pLITE201 plasmid DNA encoding the lux operon. Photoncollection was carried out for one minute under the low light imager.Twenty minutes after bacterial delivery, luminescent V. cholera wereobserved in the liver (A). Forty-eight hours after injection, lightemission was noted in the primary breast tumor in the right breast areaand a metastatic tumor (arrow) in the left breast area, and in theincision wound (B). At five days, the light emission was visible only inthe tumor regions, and non at the wound (C). Eight days after bacterialinjection, the luminescent activity was abolished from the smaller tumorregion but remained strong in the primary breast tumor (D). Homing in ofE. coli onto breast tumors in nude mice was also observed two days afterintravenous bacterial injection (E: side view, F: ventral view).

FIG. 11: Homing in of Bacteria Onto Bladder Tumors in C57 Mice

C57 mice were intravenously injected with 10⁷ attenuated V. choleratransformed with pLITE201 encoding the lux operon. Nine days afterbacterial delivery, luminescence was noted in the bladder region of thewhole animal (A). The animal was sacrificed and an abdominal incisionwas made to expose the bladder. The light emission was limited to thebladder region (B). With the removal of the bladder (C) from the mouse,the entire source of light emission was removed (D) as demonstrated bythe overlay of the low light photon emission image over the photographicimage of the excised bladder.

FIG. 12: Homing in of Bacteria onto Brain Glioma Tumors in Lewis Rats

Lewis rats were intravenously injected with 10⁸ cells of attenuated V.cholera transformed with pLITE201 encoding the lux operon. Twenty-fourhours after bacterial injection, faint luminescence was noted in thehead region of the whole animal during visualization under the low lightimager. The animals were sacrificed and their brain removed. Photoncollection was carried out for one minute from rats with (A) and without(B) brain tumors. Strong luminescence was confirmed in regions of thebrain of the rats with the brain tumor (marked with arrows in A).Luminescence was completely absent in the control brain tissues (B).

FIG. 13: Transformed Human Fibrosarcoma Cells Home in on SubcutaneousGlioma Tumors in Nude Mice

Nude mice with human breast tumors were injected intravenously with5×10⁵ human fibrosarcoma cells, which were permanently transformed withretrovirus derived from pLEIN. Seven days post-injection, the animalswere anesthetized and monitored under a fluorescent stereomicroscope.Fluorescent cells were noted only in the tumor region of the whole micethrough the skin (A1-3). Upon exposure of the tumor tissues byreflection of the overlying skin (B1-3), and in cross sections of thetumors (C1-3), fluorescent patches were visible in distinct regions.Close examination of the organs of the mice showed the presence of smallclusters of fluorescent cells in the lungs of the animals, demonstratingthe affinity of the fibrosarcoma cells for the lungs in addition to thetumorous tissues (D1-3). (Bars=5 mm (A1-C3),=1 mm (D1-D3)).

FIG. 14: Homing of Attenuated Listeria monocytogenes into SubcutaneousProstate Tumors

Nude mice with subcutaneous human PC3 prostate tumor in the right hindleg were intravenously injected with 10⁷ attenuated L. monocytogenestransformed with pSOD-gfp plasmid DNA carrying the gfp cDNA, GFPfluorescence was observed under a fluorescence stereo microscope.Twenty-seven hours after bacterial injection, GFP signal was detectedonly in the tumor region. The tumor is shown in a set of visible light(a), fluorescent (b), and visible and fluorescent light overlay (C)images. (Bars=5 mm.)

The present invention is explained by the examples.

EXAMPLE 1 Materials and Methods

(A) Bacterium strains. The bacterial strains used were attenuatedSalmonella typhimurium (SL7207 hisG46, DEL407[aroA544::Tn10]),attenuated Vibrio cholerae (Bengal 2 Serotyp 0139, M010 DattRS1), andattenuated Listeria monocytogenes (D2 mpl, actA, plcB). The bacterialstrains were kindly provided by Prof. W. Gobel (University of Wurzburg,Germany).

(B) Plasmid constructs. The plasmid pLITE201 containing the luxCDABEcassette was obtained from (Voisey and Marincs, Biotech 24, 1998,56-58). The plasmid pXylA-dual with the operon sequence of gfp-cDNA, luxAB, lux CD, and lux E under the control of the Xylose promoter waskindly provided by Dr. Phil Hill (University of Nottingham, UK).

(C) Transformation of Bacteria

The bacteria were transformed by electroporation.

(D) Tumor Cell lines. The rat C6 nitrosourea-induced glioma cell line(ATCC, Rockville, Md.) was cultured in RPMI-1640 medium (Cellgro®,Mediatech, Inc., Herndon, Va.) supplemented with 10% (v/v) FBS and 1×penicillin/streptomycin. The human PC3 prostate carcinoma cell line(ATCC, Rockville, Md.) and the Human MB-49 bladder tumor cells and rat 9L glioma cells were maintained in DMEM medium (Cellgro®, Mediatech,Inc., Herndon, Va.) supplemented with L-glutamine and 10% (v/v) FBS.HT1080 fibrosarcoma cells (ATCC, Manassas, Va.) were cultured in F12minimal essential media (Cellgro®), Mediatech, Inc., Herndon, Va.)supplemented with 10% FBS and 1× penicillin/streptomycin. The MCF-7human mammary carcinoma cell line (ATCC, Rockville, Md.), permanentlytransformed with a plasmid carrying pro-IGF-II cDNA (obtained from Dr.Daisy De Leon, Loma Linda University, Loma Linda, Calif.) was culturedin DMEM/F12 medium supplemented with 5% FBS and 560 μg/ml of G418 (LifeTechnologies, Grand Island, N.Y.).

(E) Production and propagation of retrovirus to generate alight-emitting stably transformed cell line. PT67 packing cells(Clontech, Palo Alto, Calif.) were cultured in DMEM medium supplementedwith 10% (v/v) FBS. At 70% confluence, PT67 cells were transformed withpLEIN (Clontech, Palo Alto, Calif.) using calcium phosphateprecipitation method (Profection Mammalian Transfection Systems,Promega, Madison, Wis.) for 12 hours. Fresh medium was replenished atthis time. Retroviral supernatant collected from PT67 cells 48 hourspost transformation were filtered through a 0.45 μm filter and was addedto target HT1080 cells along with polybrene to a final concentration of4 μg/ml. The medium was replaced after 24 hours and the cells weretreated with G418 selection at 400 μg/ml and stepwise increased to 1200μg/ml.

(F) Recipient animals and tumor models. Five- to six-week-old maleBALB/c athymic nu/nu mice (25-30 g in body weight) and Lewis rats(250-300 g in body weight) were purchased from Harlan (Frederick, Md.).C57BL/6J Min/+ mice were obtained from Jackson Laboratories (Bar Harbor,Me.), Min (multiple intestinal neoplasia) is an autosomal dominant traitinvolving a nonsense mutation in codon 850 of the murine Apc gene, whichrenders these animals susceptible to spontaneous intestinal adenomaformation. Female BALB/c athymic nu/nu mice bearing MCF-7 human breasttumor implants were generated and kindly provided by Dr. Daisy DeLeonand Dr. Tian (Loma Linda University, Loma Linda, Calif.). C57 mice withorthotopically implanted human MB-49 tumor cells in the bladder weregenerated and kindly provided by Dr. Istvan Fodor (Loma LindaUniversity, Loma Linda, Calif.). All animal experiments were carried outin accordance with protocol approved by the Loma Linda University animalresearch committee. The animals containing recombinant DNA materials andattenuated pathogens were kept in Loma Linda University animal carefacility under biosafety level two.

(G) Propagation of recombinant vaccinia Virus. Vaccinia virus Listerstrain (LIVP) was used as a wild type virus. Recombinant Vaccinia virusrVV-ruc-gfp was constructed by inserting, via homologous recombination,the ruc-gfp-cassette into the Vaccinia virus genome (Wang et al., Proc.Biolumin. Chemilumin. 9, 1996, 419-422). The virus was amplified in CV-1cells by addition of virus particles at a multiplicity of infection(MOI) of 0.1 pfu/cell to CV-1 cell monolayers followed by incubation at37° C. for 1 h with brief agitation every 10 min. At this time, thesupernatant fluid with virus particles was removed, and the cellmonolayers were washed once with serum free medium. Complete growthmedium was then added and the cells were incubated at 37° C. rVV-ruc-gfpvirions propagated in CV-1 cells were purified through a sucrosegradient. A plaque assay was used 72 h after infection to determine thetiter of recombinant virus by staining the cells with 50% crystal violetsolution in ethanol.

(H) Generation of mice carrying tumor implants. To obtain tumors in nudemice, C6 glioma cells were grown, harvested and the cell number wasdetermined by the Trypan Blue exclusion method. Disinfectant was appliedto the skin surface, then approximately 5×10⁵ cells were suspended in100 μl of phosphate buffered saline (PBS) and injected subcutaneouslyinto the right lateral thigh of each mouse. Tumor growth was monitoredby recording the size of the tumor with a digital caliper. Tumor volume(mm³) was estimated by the formula (L×H×W)/2, where L is the length, Wis the width, and H is the height of the tumor in mm.

Intracerebral glioma tumors were generated by injecting C6 glioma cellsinto the head of rats. Prior to injection, rats were anesthetized withsodium pentobarbital (Nembutal® Sodium solution, Abbot Laboratories,North Chicago, Ill.; 60 mg/kg body weight). A midline scalp incision(0.5-1 cm) was made, the skin was retracted, and a 1 mm burr hole wasmade in the skull at a location 2 mm to the left and 2.5 mm posterior tothe brigma. Tumor cells were pipetted into an insulin syringe, which wasfitted with a 29-gauge needle and mounted in a stereotactic holder. Theneedle was inserted vertically through the burr hole to a depth of 3 mm.After injection into the brain of 5×10⁵ C6 cells in a 10 μl volume, theneedle was kept in place for 15 sec and then withdrawn. The skinincision was closed with surgical clips. Mice bearing subcutaneousprostate tumors were generated over a period of one month followingsubcutaneous implantation of 3×10⁶ PC3 human prostate cells.

MB-49 human bladder tumor cells were implanted in the C57 mouse bladderto produce animals with bladder tumors. To generate animals with breastcancer (Tian and DeLeon, submitted for publication), female nude micewere first implanted with 0.72 mg/90 day-release 17β-estradiol pellets(Innovative Research, Rockville, Md.) in the skin to facilitate breasttumor development and metastasis. One day after estrogen pelletimplantation, 1×10⁶ MCF-7 human breast carcinoma cells transformed withpro-IGF-II (Dull et al., Nature 310 (1984), 777-781) were implanted inthe mammary fat pad. For orthotopic transplants, tumors developed fromimplanted cells were resected and minced into 1-mm³ cubes for tissuetransplantation into the mammary fat pad.

(I) Assay of Renilla luciferase in live animals. Mice were anesthetizedwith Nembutal (60 mg/kg body weight) before every Renilla luciferaseassay. Renilla luciferase activities were determined after intravenousinjection of a mixture of 5 μl of coelenterazine (0.5 μg/μl dilutedethanol solution) and 95 μl of luciferase assay buffer (0.5 M NaCl; 1 mMEDTA; and 0.1 M potassium phosphate, pH 7.4). Whole live animals werethen imaged in a dark box using a Hamamatsu low light video camera, andthe images were recorded using Image Pro Plus 3.1 software (MediaCybernetics, Silver Spring, Md.). The pseudocolored photon emissionimage was superimposed onto the gray scale image of the animal in orderto precisely locate the site of light emission.

(J) Fluorescence microscopy of live animals. Mice were anesthesized withNembutal (60 mg/kg body weight) before tumor visualization. Externalimaging of GFP expression in live animals was performed using a LeicaMZ8 stereo fluorescence microscope equipped with a mercury lamp powersupply and a GFP filter (excitation at 470 nm). Images were capturedusing a SONY DKC-5000 3CCD digital photo camera.

(K) Detection of luminescence and fluorescence. Immediately beforeimaging, mice and rats were anesthetized with Nembutal® (60 mg/kg bodyweight). The animals were placed inside the dark box for photon countingand recording superimposed images (ARGUS 100, Hamamatsu, Hamamatsu,Japan). Photon collection was for one minute from ventral and dorsalviews of the animals. A light image was then recorded and the low lightimage was then superimposed over the light image to record the locationof luminescent activity.

Imaging of GFP expression in tumors of live animals was performed usinga Leica MZ8 stereo fluorescence microscope equipped with a mercury lamppower supply and a GFP filter (excitation at 470 run). Images werecaptured using a SONY DKC-5000 3CCD digital photo camera.

(L) Histology of tumor tissues. Under anesthesia, the animals wereeuthanized with an overdose of Nembutal®. The tissues of interest wereremoved, embedded in Tissue-Tek OCT compound (Miles Scientific,Naperville, Ill.) and immediately frozen in liquid nitrogen withoutfixation. Frozen sections were cut at −20° C. using a Reichert-Jung Cyocut 1800 cryostat. GFP fluorescence of the tissues was monitored under aLeica fluorescence microscope and the images were recorded usingPhotoshop software.

EXAMPLE 2 Results Obtained by Intravenous Injection of RecombinantVaccinia Virus rVV-ruc-gfp into Mice

(A) Monitoring of Virus-Mediated Marker Gene Expression inImmunodeficient Mice

Vaccinia virus (1×10⁸ pfu) carrying the Renilla luciferase-GFP fusionexpression cassette (rVV-ruc-gfp) was introduced intravenously into nudemice with no tumors. The animals were observed once every 3 days over atwo-week time period under the low-light imager to monitor luciferasecatalyzed light emission immediately after intravenous injection ofcoelenterazine, and under a fluorescence microscope to visualize GFPexpression. Neither apparent luminescence nor green fluorescence wasdetected in the animals when imaged externally, except at certainlocations that had small skin lesions. Such luminescence andfluorescence signals disappeared after a few days once the lesions hadhealed. Animals were sacrificed one week and two weeks after viralinfection, and their organs were removed and examined for the presenceof luminescence and GFP fluorescence signals. One week after viralinjection, no luminescence or green fluorescence could be detected inbrain, liver, lung, spleen, kidney or testis. These results indicatedthat the rVV-ruc-gfp virus did not show organ specificity afterinjection and that the virus seemed to be cleared from the animal by theimmune system soon after systemic delivery via the bloodstream.

(B) Visualization of Vaccinia Virus-Mediated Marker Gene Expression inGlioma Tumors of Live Nude Mice

The distribution of injected Vaccinia virus in nude mice bearingsubcutaneously implanted C6 glioma tumors was examined. Nude mice withtumors approximately 500 mm³ in size were injected intravenously with1×10⁸ pfu of the rVVruc-gfp virus. Seven days after virus injection, theanimals were monitored for GFP expression under a fluorescencemicroscope to determine the presence of viral infection andmultiplication in the tumors, which had grown to approximately 2500 mm³in size. Surprisingly, green fluorescence was detected only in the tumorregions in live animals. Seven days after viral injection, the GFPfluorescence was very intensely localized in a patch-like patternrestricted to the tumor region (FIG. 1A-A″). These patches, often seenat the end of blood vessel branches, may have indicated local viralinfection of tumor cells that surround the leaky terminals of capillaryvessels. During real-time observation of the same tumors, the GFP signalfrom the center of these patches started to disappear, and new greenfluorescent centers appeared in the form of rings at the periphery ofthe fading patches. The new sites of intense GFP fluorescence may haveresulted from progression of the viral infection to nearby cells withinthe tumor during tumor growth and expansion. After careful examinationof the mice, with the exception of the tumor region, no detectable greenfluorescence was seen elsewhere on the body surface or in the dissectedorgans. This experiment clearly showed that a mature solid tumor couldbe easily localized by the labeled Vaccinia virus, based onlight-emission, and it also demonstrated the affinity of virus particlesfor the tumor tissue.

To determine whether tumor size and vascularization are decisive factorsfor viral retention in tumors, nude mice were intravenously injectedwith 1×10⁸ rVV-ruc-gfp Vaccinia virus particles one day aftersubcutaneous C6 cell implantation. Surprisingly, 4 days after viralinjection GFP expression was seen in 5-day-old C6 tumors that had avolume of about 25 mm³ (FIG. 1B-B″). Examination of labeled Vacciniavirus tumor targeting by visualization of GFP expression in implantedtumors younger than 5 days was not feasible in live mice, sincesufficient levels of marker gene expression required approximately 4days to allow detection under a fluorescence microscope.

The finding that injection of the rVV-ruc-gfp Vaccinia virus into thebloodstream of the host resulted in GFP expression and accumulation intumors suitable for non-invasive tumor detection allowed us to followthe entry and replication process of this virus in the same animal inreal time (FIG. 1 C-C″, D-D″ and E-E″). A continuously increasing levelof GFP fluorescence was observed in the same animal throughout 20 daysfollowing viral injection, which was the time scheduled beforesacrificing the animals. Such an increase in detectable fluorescence wasindicative of a very strong viral replication in the tumor tissue, thelatter appearing to function as a protective immunoprivilegedenvironment for viral replication. Interestingly, the location of bloodvessels and the neovascularization within the periphery of the enlargingtumor were readily visible and confirmed by external visualizationagainst a bright green fluorescent background (FIG. 1A-A″, D-D″, E-E″and FIG. 2).

To determine the location of viral infection within the tumors, theanimals were sacrificed and the skin over the tumor was carefullyreflected to expose the tumor. In the exposed tumor, GFP fluorescencewas found to be concentrated exclusively in the tumor tissue (FIG. 3B-B″and D-D″). The non-tumorous thigh muscles did not show any fluorescenceof viral infection, as indicated by arrows in FIG. 3D-D″. The skinoverlying the tumor was also non-fluorescent (indicated by asterisks inFIG. 3B-B″ and D-D″). Cross sections of the tumor, however, revealedthat strong green fluorescent regions were mostly found as patches inthe periphery of the tumor (double arrows in FIG. 3C-C″) where theactively dividing tumor cells are presumably located.

To further examine the pattern of viral infection in C6 glioma tumorsbased on GFP expression, the tumor tissues were sectioned formicroscopic analysis under the fluorescence microscope. Comparativeanalysis of various tissue sections revealed that GFP fluorescence waspresent in large clusters of cells within the tumor (FIG. 4), but nofluorescence was visible in normal tissues such as the heart, lung,liver, spleen, and kidney.

In addition to GFP, the recombinant rVV-ruc-gfp virus carried a secondmarker gene, which encoded the Renilla luciferase in the form of afusion protein with GFP. Therefore we were able to directly superimposethe site of GFP fluorescence with light emission from Renilla luciferasein the tumors. Immediately after coelenterazine (substrate for Renillaluciferase) was delivered by intravenous injection, a very strongluciferase activity was recorded only in the tumor region under a lowlight video camera (FIG. 5). By lowering the sensitivity of the lowlight video camera to avoid saturation of light detection, we were ableto identify the Renilla luciferase gene expression in localized patchesin the periphery of the tumor. These patch-like patterns preciselycorrelated with the GFP signals.

(C) Affinity of Vaccinia Virus Delivered to the Bloodstream forDifferent Tumors Implanted into Animals

To determine whether the attraction of the Vaccinia virus was limited toglioma tumors or whether this attraction could be observed in othertumors, recombinant Vaccinia virus was recombinantly introduced intomice that carried different types of implanted tumors. One of thesetumor models was a nude mouse with implanted subcutaneous PC-3 humanprostate carcinoma. Although the PC3 implants from which tumorsdeveloped grew at a much slower rate than the implanted subcutaneousglioma tumors, these tumors showed the same dynamics with regards toVaccinia virus infection when identical titers (1×10⁸) were injectedintravenously (FIG. 6A-A″). Similar to our findings with glioma tumors,GFP expression was initially detected 4 days after virus injection, andthe fluorescence lasted throughout the 3-week observation period.

Female nude mice with established breast tumors were also used forlabeled Vaccinia injections. These breast tumors were allowed to growfor 6 months after the animals received implants of MCF-7 human breastcarcinoma cells transformed with pro-IGF-11 cDNA. At the time ofVaccinia virus injection, the tumors had reached maximum growth and thetumor volume (about 400-500 mm³) did not change significantly during theexperimental period. Similar to previous experiments, 6 days afterintravenous delivery of 1×10⁸ rVV-ruc-gfp virus particles, strong GFPexpression was observed in the breast tumor region (FIG. 6B-B″, FIG.7A-A″ and B-B″) and nowhere else in the body.

Examination of cross sections of virus-infected breast tumors revealedluminescent “islands” throughout the tumors without any indication ofcentral or peripheral preference of infection (FIG. 7C-C″). The MCF-7tumor cells used in these breast tumor models are known to metastasizeand in addition to the primary solid tumor, a smaller metastasized tumorfound on the left lateral side of the body showed GFP fluorescence (FIG.7D-D″, E-E″, and F-F″). Excised lung tissues were also examined fordetection of metastases. Metastasized tumors as small as 0.5 mm indiameter on the surface of the lung were positive for GFP fluorescence(FIG. 7G-G″). The presence of a strong Renilla luciferase-mediated lightemission confirmed the expression of the luciferase-GFP fusion proteinin these breast tumors but nowhere else in the body when the substratecoelenterazine was injected intravenously into the live animals. Theseexperiments showed that intravenously delivered Vaccinia virus particleswere selectively attracted to and replicated in primary and metastasizedbreast tumors in nude mice, likely as a result of the immunocompromisedstate of the tumor microenvironment.

To determine whether virus particles could move out of tumors andre-enter the circulation, we injected C6 glioma cells into the thigh ofmice to form a second tumor in animals already carrying a breast tumorinfected with labeled Vaccinia virus. If the virus particles werereleased from the tumor to re-enter the circulation in significantnumbers they would be able to colonize the newly implanted glioma tumor.Monitoring of these second tumors showed that no GFP signal was visiblein the new glioma tumor 7 and 14 days after implantation of the gliomacells. To demonstrate that the newly implanted glioma tumors could betargeted by labeled Vaccinia virus, a second dose of rVV-ruc-gfp virus(1×10⁸ pfu) was intravenously injected. Five days later, tumor-specificGFP expression was detected in the newly formed glioma tumor in additionto GFP expression seen in the original breast tumor. These findingssuggested that the virus particles in infected tumors were either notreleased back into the circulation at all, or were not released insufficient numbers to infect and replicate in a second tumor.

Two additional tumor models, including Lewis rats with intracranial C6rat glioma tumors and C57 mice with MB-49 human bladder tumors in thebladder, were used for Vaccinia injections. To determine whethertumor-affinity of virus particles is a phenomenon limited to tumors innude mice with a diminished T-lymphocyte function or whether it is ageneral protective property of tumors that may be demonstrated also inimmunocompetent animals, Lewis rats with intracranial C6 rat gliomatumors and C57 mice with MB-49 human bladder tumors in the bladder wereused. A total of 5×10⁵ C6 glioma cells in a 100 μl volume werestereotactically implanted in the brains of 2 of 4 immunocompetent Lewisrats, and the tumors were allowed to grow for 5 days. The other 2 ratswere injected intracranially with phosphate-buffered saline to serve ascontrols. On day six, all 4 rats were intravenously injected withrVV-ruc-gfp virus particles via the femoral vein. Five days after virusinjection, all 4 animals were sacrificed, and their brains werecarefully excised for analysis by fluorescence microscopy. GFPexpression was detected in the brains with implanted intracranial tumors(FIG. 6C-C″) while no GFP expression was seen in the control brains. Inparallel experiments, C57 mice, with or without bladder tumors, weredivided into two groups. One group was injected intravenously withrVV-ruc-gfp Vaccinia virus (1×10⁸ pfu) and the other with salinesolution as control. Five days after virus injection, the animals weresacrificed and examined under the fluorescence microscope. GFPexpression was observed in the bladder tumor region in C 57 mice but notin control mice (FIG. 6D-D″).

Taken together, these experiments show that Vaccinia virus particleswere selectively accumulated and retained in a variety of tumors,probably protected by the tumor microenvironment, and that they were notable to survive in the non-tumorous tissues of immunocompromised as wellas immunocompetent animals. The tumor-targeting process by intravenouslyinjected Vaccinia virus carrying the light-emitting dual marker genedemonstrated the ability of the Vaccinia virus system to detect primaryand metastatic tumors in live animals.

EXAMPLE 3 Results of Intravenous Injection of Bacterial and MammalianLight-Emitting Cells into Mice

(A) Visualization of Light Emitting Bacteria Present in Whole AnimalsAfter Intravenous Injection

To determine the fate of intravenously injected luminescent bacteria inthe animals, 10⁷ bacteria carrying the pLITE201 plasmid in 50 μl wereinjected into the left femoral vein under anesthesia. Following closureof the incision with sutures, the mice were monitored under the lowlight imager (ARGUS 100 Camera System, Hamamatsu, Hamamatsu, Japan) inreal time and photons were collected for one minute. The imaging wasrepeated in two-day time intervals to determine the presence of lightemission from a given animal. It was found that the distribution patternof light emission following an intravenous injection of bacteria intomice was characteristic of the bacterial strains used. Injection of theattenuated V. cholera into the bloodstream resulted in light emissionlocalized in the liver immediately. Injection of S. typhimurium,however, was widely disseminated throughout the body of the animalsuggesting a difference in the interaction with host cell system (FIG.8A-8D). Imaging the same animals 24 and 48 hours post-infection showedthat all of the detectable light emission from the earlier timediminished rapidly and was eliminated completely from the injectedanimal. These findings suggest that light emitting bacteria injectedinto the bloodstream via the femoral vein are cleared. This process wasconfirmed by photon emission analysis of excised organs, which werefound to lack light emission. Similar data were obtained inimmunocompetent mice and rats suggesting that the removal of bacteriafrom the blood is efficient in both systems.

(B) Bacteria Home in on Glioma Tumors in Nude Mice

To determine if bacteria preferentially colonize tumorous tissues, nudemice with ten-day-old tumors (about 500 mm³) in the tight hind leg wereinjected intravenously via the femoral vein with 10⁷ S. typhimurium or10⁷ V. cholera in a 50 μl volume of bacterial suspension. Followinginjection, the incision wounds were sutured and the animals weremonitored for six days under the low light imager. At each observationtime point, photons were collected for exactly one minute. In miceinjected with S. typhimurium, luminescent bacteria were disseminatedthroughout the whole body of the animal similar to the findings in thenon-tumorous mice (FIG. 9A). Nude mice injected with V. cholera,demonstrated luminescent activity only in the liver region during theearly observation period (FIG. 9E). Regardless of the bacterial straininjected, two days after injection, luminescent activity was observedonly in the tumor region (FIGS. 9B and 9F). Monitoring of the mice underthe low light imager on days four and six post-injection showeddecreased amounts of detectable luminescence in the tumors of animalsinjected with S. typhimurium (FIGS. 9C and 9D. This finding was inmarked contrast with the findings in the tumors of mice injected with V.cholera, which demonstrated not only survival but also propagation ofthe bacteria in the tumor mass with a dramatic increase in lightemission (FIGS. 9G and 9H).

Nude mice bearing subcutaneous human PC3 prostate tumors in the righthind leg were intravenously injected with 10⁷ attenuated L.monocytogenes transformed with pSOD-gfp plasmid DNA carrying the gfpcDNA. GFP fluorescence was observed under a fluorescencestereomicroscope. Twenty-seven hours after bacterial injection, GFPsignal was detected only in the tumor region (FIG. 14). No GFP signalwas observed in the rest of the animal.

(C) Determination of Minimum Size and Age of Glioma Tumors Necessary forBacterial Infection

The purpose of this experiment was to determine whether the size of thetumor has any influence on its ability to be colonized by bacteria.Tumors were induced in the right hind leg of nude mice by subcutaneousinjection of glioma cells as described. On days 0, 2, 4, 6, 8, and 10 oftumor induction, attenuated S. typhimurium and V. cholera with thepLITE201 plasmid were injected intravenously through the femoral vein.Presence of luminescent bacteria in the tumor was determined by photoncollection for exactly one minute under the low light imager two andfour days post-infection. The tumor volume was also determined bymeasuring the dimensions with a digital caliper. The earliest time-pointwhen luminescent activity was noted in the tumors was on day eight aftertumor induction. Corresponding tumor volumes were approximately 200 mm³.

(D) Bacteria Home in on Breast Tumors of Nude Mice

In order to determine whether colonization of tumors is limited toglioma cells or whether this is a general phenomenon observed with alltumors, female nude mice baring tumors in the right breast pad wereintravenously injected with 10⁷ V. cholera in a 50 μl volume of bacteriasuspension. The animals were monitored within the first 10 minutes afterinoculation under the low light imager for one minute and demonstratedthe typical luminescent pattern in the liver region (FIG. 10A). Two dayslater, while the liver had become clear of luminescent bacteria, thebreast tumor was colonized by the labeled V. cholera. In addition to themain tumor, a metastatic tumor in the left breast demonstratedluminescent activity. (FIG. 10B). On day five, the animals had clearedthe bacteria that colonized the incision wound, however, the tumorsremained luminescent (FIG. 10C). FIG. 10D shows the continuedcolonization and propagation of the bacteria in the main tumor, whilethe metastatic, smaller tumor had become cleared. Luminescent activitycontinued for over 45 days in the right breast tumor. Similarexperiments were conducted using E. coli to demonstrate that homing inof tumors by bacteria is not strain dependent (FIGS. 11E and 11F).(FIGS. 10E and 10F).

To determine whether the bacteria from the tumor enter the bloodcirculation in significant quantities to colonize other sites, a secondtumor (C6 glioma) was induced in these animals in the right hind led.The tumor was allowed to grow for 10 days. No luminescent activity wasobserved in the glioma tumor demonstrating the absence of a significantbacteria that would cause colonization of this tumor. However, when theanimal was rechallenged with 10⁷ attenuated V. cholera intravenously,the leg tumor showed strong luminescent activity.

The findings of these experiments demonstrate that larger tumors retainbacteria more effectively over time. Furthermore, the bacteria withinthe tumors do not escape into the blood in sufficient quantities toinfect susceptible sites such as other tumors.

(E) Bacteria Home in on Bladder Tumors in Immunocompetent Mice

C57 mice were intravenously injected with 10⁷ attenuated V. choleratransformed with pLITE201 encoding the lux operon. On day nine afterbacterial delivery, luminescent activity was recorded by photoncollection for one minute under the low light imager. Light emission wasnoted in the bladder region of the whole animal (FIG. 12A). (FIG. 11A).The animals were sacrificed and an abdominal incision was made to exposethe bladder. Luminescent activity was positively confirmed to be limitedto the bladder (FIG. 11B). Upon removal of the bladder from the mice,luminescent activity was no longer visible anywhere in the animals,however, the excised bladders continued to demonstrate light emission.(FIG. 11C). Based on the results of this experiment, bacteria can targettumors in immunocompetent as well as nude mice. Furthermore, thebacteria can also target smaller tumors.

(F) Bacteria Home in on Glioma Tumors in the Brain of Rats

Lewis rats with glioma tumors in the brain were intravenously injectedwith 10⁸ attenuated V. cholera with the pLITE201 plasmid through theleft femoral vein to determine if bacteria can cross the blood-brainbarrier and target tumors in immunocompetent animals. The whole animalswere monitored for one minute under the low light imager the followingday and low levels of luminescent activity was observed through theskull. The rats were sacrificed and the brain tissue was removed in onepiece in order to further evaluate the exact location of the luminescentbacteria. Visualization of the excised brain under the imagerdemonstrated strong luminescent activity in specific regions of thebrain. (FIG. 12A). Similar imaging of control rats without brainstumors, which were intravenously injected with the labeled bacteria,demonstrated absence of any luminescent activity (FIG. (FIG. 12B).

(G) Transformed Human Fibrosarcoma Cells Home in on Subcutaneous GliomaTumors in Nude Mice

Nude mice with human breast tumors were injected intravenously with5×10⁵ human fibrosarcoma cells, which were permanently transformed withretrovirus derived from pLEIN. Seven days post-injection, the animalswere anesthetized with Nembutal, and monitored under a fluorescentstereomicroscope. Fluorescent cells were noted only in the tumor regionof the whole mice through the skin (FIG. 10A1-3). Upon exposure of thetumor tissues by reflection of the overlying skin (FIG. 10B1-3), and incross sections of the tumors, (FIG. 10C1-3), fluorescent patches werevisible in distinct regions. Close examination of the organs of the miceshowed the presence of small clusters of fluorescent cells in the lungsof the animals, demonstrating the affinity of the fibrosarcoma cells forthe lungs in addition to the tumorous tissue.

EXAMPLE 4 Construction of Bacterial Plasmid Vectors That Carry theLight-Emitting Protein Encoding Expression Cassettes and the TherapeuticGene Expression Constructs in Cis Configuration

(A) Rationale

Using the light-emitting expression systems described above, tumorscould be imaged based on light emission for up to 45 days in animals.These findings suggest a remarkable plasmid DNA stability in bacteria inthe absence of selection. Therefore, by placing the therapeutic genecassette in cis configuration with the light-emitting protein expressioncassette on the same replicon, light emission can be used as anindicator of therapeutic construct presence and stability.

In contrast to light-emitting proteins, the therapeutic proteins,endostatin and Pseudomonas exotoxin/TGF alpha fusion protein, arerequired to be-secreted from the bacteria into the medium or into thecytosol of tumor cells for inhibition of tumor growth. To achieveprotein secretion from the extracellularly replicating E. coli cellsinto the tumor, two constructs with different signal sequences can bedesigned. For secretion of endostatin, the ompF signal sequence can beplaced upstream of the coding sequence of endostatin, which facilitatesthe secretion into the periplasmic space. To release the endostatin intothe medium, an additional protein, the PAS protein, needs to becoexpressed with endostatin. PAS has been shown to cause membraneleakiness and the release of secreted proteins into the medium (Tokugawaet al., J. Biotechnol. 37 (1994), 33; Tokugawa et al., J. Biotechnol. 35(1994), 69). The second construct for the secretion of Pseudomonasexotoxin/TGF alpha fusion protein from E. coli has the OmpA signalsequence upstream of the fusion gene and the release from theperiplasmic space into the medium is facilitated by sequences present indomain II of the exotoxin (Chaudhary et al., PNAS 85 (1988), 2939; Kondoet al., J. Biol. Chem. 263 (1988), 9470; Kihara and Pastan, Bioconj.Chem. 5 (1994), 532). To promote secretion of endostatin and Pseudomonasexotoxin/TGF alpha fusion protein from L. monocytogenenes, the signalsequence of listeriolysin (LLO) (Mengaud et al., Infect. Immun. 56(1988), 766) can be placed upstream of each coding sequence.

For regulation of endostatin and Pseudomonas exotoxin/TGF alpha fusionprotein expression levels in bacteria, vectors can be generated wherethe therapeutic protein encoding genes are under the control of the T7promoter or the P_(spac) synthetic promoter (Freitag and Jacobs, Infect.Immun. 67 (1999), 1844). Without exogenous induction, the levels of thetherapeutic proteins are low in E. coli and in L. monocytogenes. Theminimal levels of therapeutic proteins in bacteria provide greatersafety following intravenous injection of the engineered bacteria. Inthe following, six newly constructed plasmid DNAs for constitutive andregulated expression of endostatin and Pseudomonas exotoxin/TGF alphafusion protein in E. coli and L. monocytogenes are described. Allplasmids to be transferred into E. coli will carry the constitutivelyexpressed bacterial lux operon, and all the plasmids to be transferredinto L. monocytogenes will carry the constitutively expressed sod-gfpcassette. Plasmids BSPT#1-ESi and BSPT#2-Pti are able to replicate in E.coli only, and plasmids BSPT#3, #4, #5, and #6 replicate in E. coli andL. monocytogenes.

(B) Construction of Plasmid Vectors for Protein Expression and Secretionfrom E. coli

The construction of the endostatin secretion vector to be used in E.coli is as follows. The coding sequence of human endostatin (591 bp)will be amplified by PCR from the plasmid pES3 with the introduction ofthe required restriction sites on both ends, followed by ligation into apBluescript (Clontech Corp., USA) cloning vector to generate pBlue-ES.The ompF signal sequence (Nagahari et al., EMBO J. 4 (1985), 3589) isamplified with Taq polymerase and inserted upstream in frame with theendostatin sequence to generate pBlue-ompF/ES. The expression cassettedriven by the T7 promoter will be excised, and inserted into thepLITE201 vector described in Example 1(B), above, carrying the luxCDABEcassette, to produce the plasmid pLITE-ompF/ES. The sequence encodingthe PAS factor (a 76 amino acid polypeptide) will be amplified from thechromosomal DNA of Vibrio alginolyticus (formerly named Achromobacteriophagus) (NCIB 11038) with Taq polymerase using the primers5′-GGGAAAGACATGAAACGCTTA3-′(SEQ ID NO: 1) and5′-AAACAACGAGTGAATTAGCGCT-3′ (SEQ ID NO: 2), and inserted into themultiple cloning sites of pCR-Blunt (Clontech Corp., USA) to create theexpression cassette under the control of the lac promoter. The resultingplasmid will be named pCR-PAS. The lac promoter linked to the pas genewill be excised from pCR-PAS and inserted into pLITEompF/ES to yield thefinal plasmid BSPT#1-ESI.

Plasmid pVC85 (Pastan, see above) contains a T7 promoter, followed by anompA signal sequence, and a sequence encoding domain II and III ofPseudomonas exotoxin (PE40). The DNA sequence encoding PE40 will beexcised with restriction enzymes and replaced with a fragment ofPE37/TGF alpha (Pseudomonas exotoxin A 280-613/TGF alpha) obtained fromthe plasmid CT4 (Pastan, see above) to create the plasmid pVC85-PE37/TGFalpha. The expression cassette of ompAPE37/TGF alpha linked to the T7promoter will be excised and inserted into pLITE201 to yield the finalplasmid BSPT#2-PTI.

(C) Construction of Plasmid Vectors for Protein Expression and SecretionFrom L. monocytogenes

Genes encoding endostatin or PE37/TGF alpha will be inserted downstreamof the listeriolysin (LLO) signal sequence in the plasmid pCHHI togenerate pCHHI-ES and pCCHI-PE37/TGF alpha. Constitutive expression ofthe therapeutic proteins will be obtained by linking the above secretioncassettes to the listeriolysin promoter obtained from the pCHHI vector.The SOD-GFP expression cassette, excised from the plasmid pSOD-GFP (Gotzet al. PNAS in press.) will be inserted into pCHHI-ES to generateBSPT#3-ESc, and into pCCHI-PE37/TGF alpha to generate BSPT#4-PTc. Forthe expression of the therapeutic proteins under the control of an IPTGinducible promoter, the listeriolysin promoter in BSPT#3-ESc andBSPT#4-PTc will be replaced with the P_(spac) promoter from the plasmidpSPAC (Yansura and Henner, PNAS USA 81 (1984), 439) to generateBSPT#5-ESi and BSPT#6-PTi. P_(spac) is a hybrid promoter consisting ofthe Bacillus subtilis bacteriophage SPO-1 promoter and the lac operator.IPTG-induced GFP expression from the P_(spac) promoter has beendocumented in L. monocytogenes in the cytosol of mammalian cells.

EXAMPLE 5 Demonstration of the Expression of Luciferase and GFP inBacteria and Verification of the Secretion of Endostatin and RecombinantToxin/TGF Alpha Fusion Protein and their Function in Cell Culture Assays

To be able to detect the presence of E. coli and L. monocytogenes intumor tissues in live animals, the levels of the constitutivelyexpressed luciferase and GFP in bacteria need to be adequate. Therefore,after transformation of recipient E. coli or L. monocytogenes with theconstructs described in Example 4, the colonies with the highestluciferase light emission or OFP fluorescence will be selected. Inaddition to characterizing light emission from each selected colonybefore intravenous injection, the ability of the selected transformantsto secret endostatin and Pseudomonas exotoxin/TGF alpha fusion proteininto the medium needs to be confirmed. The presence of endostatin andPseudomonas exotoxin/TGF alpha fusion protein synthesized within E. coliand L. monocytogenes will be determined by extracting these proteinsfrom the cell pellet. The secreted proteins in the medium will beconcentrated and analyzed by gel separation and the quantity will bedetermined by Western blotting. It is imperative to determine thepercentage of the newly synthesized proteins expressed from each plasmidconstruct in either E. coli or L. monocytogenes that is present in themedium. It is also essential to confirm, in addition to constitutiveexpression of endostatin and Pseudomonas exotoxin/TGF alpha fusionprotein, that expression can be induced in E. coli and in L.monocytogenes upon the addition of IPTG to the bacterial culture medium.For the design of future tumor therapy protocols, the relative amountsof protein secreted by the constitutive expression system needs to becompared to the induced expression levels for a defined time periodfirst in bacterial cultures. It is equally essential to determine thatboth proteins when synthesized in E. coli and L. monocytogenes arebiologically active if generated from the proposed constructs. Bothproteins were synthesized previously in E. coli and were shown to beactive.

The results of the experiments described below should confirm whetherendostatin is successfully secreted from E. coli using the OmpF signalpeptide in combination with PAS pore forming protein expression. Theseexperiments will also show if the PE40/TGF alpha and PE37/TGF alphafusion proteins are secreted from bacteria using the OmpA signal peptidein combination with domain II of PE. Further, the listeriolysin signalpeptide may also facilitate the secretion of endostatin and the chimerictoxin/TGF alpha fusion protein into the medium as well as into thecytosol of infected tumor cells. Using the migration inhibition assayand the protein synthesis inhibition assay, it can be expected todetermine that both proteins secreted into the medium are biologicallyactive. The presence and quantities of these proteins may be regulatedby replacing the constitutive promoters with promoters that can beinduced by IPTG.

In addition to the secretion system described below, alternativesecretion systems such as the E. coli HlyBD-dependent secretion pathway(Schlor et al., Mol. Gen. Genet. 256 (1997), 306), may be useful.Alternative secretion signals from other gram positive bacteria, such asthe Bacillus sp. endoxylanase signal peptide (Choi et al., Appl.Microbiol. Biotechnol. 53 (2000), 640; Jeong and Lee, Biotechnol.Bioeng. 67 (2000), 398) can be introduced.

(A) Confirmation of Endostatin and Pseudomonas Exotoxin/TGF Alpha FusionProtein Secretion from Bacteria into Growth Medium

E. coli strains (DH5α and BL21(λDE3) will be transformed with BSPT#1-ESiand BSPT#2-PTi plasmid DNA. L. monocytogenes strain EGDA2 will betransformed with plasmids BSPT#3-ESc, BSPT#4-PTc, BSPT#5-ESi, andBSPT#6-PTi individually. After plating on appropriateantibiotic-containing plates, individual colonies will be selected fromeach transformation mixture. These colonies will be screened under a lowlight imager and fluorescence microscope for luciferase and GFPexpression, respectively. Three colonies with the most intense lightemission from each transformation batch will be chosen for furtherstudies. To verify the secretion of endostatin and Pseudomasexotoxin/TGF alpha fusion protein from each selected transformant, thecells will be grown in minimal medium to log phase. After centrifugingdown the bacteria, the supernatants will be passed through a0.45-μm-pore-size filter, and the bacterium-free medium will be used forprecipitation of the secreted proteins. The precipitates will becollected by centrifugation. Pellets will be washed, dried, andre-suspended in sample buffer for protein gel separation. Proteins fromaliquots corresponding to 10 μl of bacterial culture will be compared toproteins from 200 μl of culture supernatant after separation in a 10%SDS-polyacrylamide gel. Western blot analysis will be performed usingpolyclonal antibody against endostatin (following the antibodyproduction protocol described by Timpl, Methods Enzymol. 82 (1982), 472)and monoclonal antibody against TGF alpha (oncogene Research Products,Cambridge, Mass., USA). The optimal growth conditions will beestablished for secretion by sampling the growth medium at differenttimes during growth. A similar method has been used previously toanalyze secreted proteins in Salmonella typhimurium culture supernatant(Kaniga et al., J. Bacteriol. 177 (1995), 3965). By use of these methodsthe amount of secreted proteins in the bacterial culture mediumgenerated by each of the constructs without induction will beestablished. To estimate the increase in the amount of secreted proteinsin the medium, IPTG-dependent promoter activation experiments will becarried out by adding IPTG to the bacterial culture in log phase for 3to 6 hours, and the secreted proteins will be assayed as above.

(B) Verification of the Biological Activity of Endostatin Secreted by E.coli and L. monocytogenes Using a Migration Inhibition Assay

It has been shown that endostatin inhibits vascular endothelial growthfactor (VEGF)-induced human umbilical vein endothelial cell (HUVEC)migration. Thus, the biological activity of endostatin secreted bybacteria can be tested using the HUVEC migration assay provided byCascade Biologics, Portland, Oreg. The inhibition of cell migration willbe assessed in 48-well chemotaxis chambers (Neuro Probe, Gaithersburg,Md.) (Polyerine et al., Methods Enzymol. 198 (1991), 440).Bacterium-free supernatant from each secretion construct will be addedto HUVECs for preincubation for 30 min. After incubation, the HUVECswill be placed in the upper chamber. The migration of HUVECs into thelower chamber induced by VEGF₁₆₅ (R&D Systems, Minneapolis, Minn.) willbe quantified by microscopic analysis. The concentration of functionalendostatin in the medium will be directly proportional to the degree ofinhibition of HUVEC migration.

(C) Testing the Cytotoxic Activity of Secreted Recombinant PE ToxinTumor in Tumor Cell Cultures

The inhibitory activity of the chimeric toxin in mammalian cells will bemeasured based on inhibition of de novo protein synthesis byinactivating EF-2 (Carroll and Collier, J. Biol. Chem. 262 (1987),8707). Aliquots of bacterium-free supernatants obtained from theexpression of various recombinant PE secretion constructs in E. coli andin L. monocytogenes will be added to the C6 glioma cells or to HCTI 16colon carcinoma cells. Following treatment with medium, the mammaliancells will be pulsed with [³H]-leucine, and the incorporation will bedetermined in the protein fraction. To determine the presence ofsecreted chimeric toxin proteins in L. monocytogenes-infected mammaliancells, the bacteria will be eliminated from the medium by gentamicintreatment. The mammalian cells containing L. monocytogenes in thecytosol will be lysed, and the released bacteria removed from the lysateby filtration. The mammalian cell lysate containing the secretedchimeric toxins will be assayed in protein synthesis inhibitionexperiments. The inhibition of [³H]-leucine incorporation in tumor cellculture will be directly proportional to the amount of the biologicallyactive chimeric toxin protein in the medium and cell lysate.

EXAMPLE 6 Determination of the Entry, Localization and Distribution ofIntravenously Injected Bacteria in Tumors of Live Animals

(A) Rationale

Since only a small number of intravenously injected bacteria escape theimmune system by entering the tumor, their immediate localization is notpossible due to limited light emission in live animals. Their locationcan only be verified by sectioning the tumor to identify the earlycenters of light emission. Looking at sections at a later time point,bacteria can be seen throughout the entire tumor due to rapidreplication. To determine whether one or multiple bacteria enter throughthe same site, red fluorescent protein can be used to label theextracellularly replicating E. coli and green fluorescent protein forthe intracellularly replicating L. monocytogenes. By visualizing thedistribution of the red and green fluorescence in tissue sections, theentry sites as well as replication and localization of E. coli and L.monocytogenes can be determined individually and simultaneously in thecentral or peripheral regions of the tumor. It can be expected that thepatterns of entry and distribution obtained in implanted tumors mimicthose of spontaneous tumors, accordingly, the bacterium-based diagnosisand protein therapy will become a valid approach.

With the experiments described in section (B), below, the entry,replication, and distribution of light-emitting bacteria in spontaneoustumors can be compared to the distribution patterns in implanted tumors.Further, double-labeling experiments will allow the operator toprecisely locate the extracellularly replicating E. coli and theintracellularly replicating L. monocytogenes in the same tumor sections.Lastly, it can be determined (subsequent to a five-day bacterialcolonization) whether bacteria are distributed evenly in the tumors orpreferential localization occurs in the periphery of the tumor or in thenecrotic center. A possible reduction in bacterial entry intospontaneously occurring tumors due to the immunocompetence of theseanimals can be overcome by increasing the number of intravenouslyinjected bacteria.

(B) Intravenous Injection of E. coli Expressing Red Fluorescent Proteinand L. monocytogenes Expressing Green Fluorescent Protein into Nude Miceand into Rodents with Implanted and Spontaneous Tumors

E. coli (DH5α) carrying the DsRed (Matz et al., Nat. Biotech. 17 (1999),969) expression cassette under the control of a constitutive promoterare used in this experiment. L. monocytogenes EGD strain derivativeswith in-frame deletion in each of the virulence genes were individuallylabeled with the green fluorescent protein cassette driven by theconstitutive SOD promoter.

The localization and intratumoral distribution of bacteria will first bestudied in nude mice with implanted C6 glioma or HCT116 colon carcinomatumors. C6 glioma or HCT116 colon carcinoma cells (5×10⁵ in 100 μl) willbe subcutaneously injected into the right hind leg of the animals.Twelve days after tumor cell injection, the animals will beanesthetized, and the left femoral vein surgically exposed.Light-emitting bacteria (1×10⁶ cells re-suspended in 50 μl of saline)will be intravenously injected, and the wound incision will be closedwith sutures. Tumors will be measured three times a week using acaliper. Tumor volume will be calculated as follows: smalldiameter×large diameter×height/2.

Intracerebral glioma tumors will be generated by injecting C6 gliomacells into the head of Wistar rats. Rats will be anesthetized withKetamine (70-100 mg/kg body weight) and Xylazine (8-10 mg/kg bodyweight). A midline scalp incision (0.5-1 cm) will be made, skin will bereflected, and a 1 mm burr hole will be made in the skull located 2 mmto the left and 2.5 mm posterior to the brigma. Tumor cells will bepipetted into an insulin syringe fitted with a 29-gauge needle andmounted in a stereotactic holder. The needle will be inserted verticallythrough the burr hole to a depth of 3 mm. After injection into the brainof a 5 μl volume of either 5×10⁵ C6 cells or PBS as control, the needlewill be kept in place for 15 sec and then withdrawn. The skin incisionwill be closed with surgical clips. Ten days after cell injection, anintracranial glioma will develop which is 5-10 mm in diameter. The sameprotocols involving intravenous injection of bacteria into animals withtumors will be followed through the reminder of the proposal.

The localization of bacteria in the tumor, based on GFP or RFP, willalso be analyzed using cryosectioned tumor tissues. A reliablemorphological and histological preservation, and reproducible GFP or RFPdetection may be obtained using frozen sections after a slow tissuefreezing protocol (Shariatmadari et al., Biotechniques 30 (2001), 1282).Briefly, tumor tissues will be removed from the sacrificed animals to aPetri dish containing PBS and dissected into the desired size. Thesamples will be mixed for 2 h in 4% paraformaldehyde (PFA) in PBS atroom temperature. They will be washed once with PBS, and embedded inTissue-Tek at room temperature, and then kept in the dark at 4° C. for24 h and slowly frozen at −70° C. Before sectioning, the tissue will bekept at −20° C. for 30 min. Then, 10- to 50-μm-thick sections will becut with a Reichert-Jung Cryocut 1800 cryostat and collected onpoly-L-lysine (1%)-treated microscope slides. During sectioning, thematerial will be kept at room temperature to avoid several freezing andthawing cycles. Finally, the sections will be rinsed in PBS and mountedin PBS and kept in the dark at 4° C.

To monitor the entry of light emitting E. coli and L. monocytogenes fromthe blood stream into the tumor, 27 nude mice will be injected with C6tumor cells, and 27 nude mice with HCT116 colon carcinoma cells. Twelvedays after tumor development, 9 animals from the C6 group and 9 from theHCT116 group will receive an intravenous injection of E. coli with theRFP construct. Another 9 animals from each group will receive anintravenous injection of L. monocytogenes transformed with the GFPconstruct. The third group of 9 animals from each tumor model willreceive both E. coli and L. monocytogenes (1×10⁶ cells of each). Fivehours, 25 hours, and 5 days after injection, three animals of eachtreatment group will be sacrificed, their tumors excised, and processedindividually as described in the above cryosectioning protocol. Afterfreezing, each tumor will be cut into two halves. One half of the tumorwill be used for preparing thick sections (60-75 μm), which will beanalyzed under a fluorescence stereomicroscope to observe thedistribution of bacteria in the sections of tumors obtained from eachtime point of the experiment. The regions of interest will beidentified, thin sectioned, prepared, and analyzed with laser scanningcytometry and under the confocal microscope followed by imagereconstruction.

In parallel experiments, animals with spontaneous tumors, as listed inTable 1, will be obtained and used in intravenous injection experimentswith E. coli carrying the bacterial lux operon. Two animals of eachtumor model will be used, and the luciferase light emission monitoreddaily under the low light imager. It is expected that the spontaneouslyoccurring tumors can be imaged similarly to the implanted tumors basedon bacterial luciferase expression. Two of the spontaneous tumor models,mice with adenocarcinoma of the large intestine and mice withadenocarcinoma of the mammary tissue, will be used for bacteriallocalization experiments following intravenous injection of E. coliexpressing RFP and L. monocytogenes expressing GFP as described above.It can be expected that these experiments will emphasize thesignificance of the bacterium-based diagnosis and protein therapysystem.

TABLE 1 Spontaneous tumor animal models Animal Strain Tumor species namedescription Source References Mouse 129/Sv- spontaneous Jackson Zhu etal., Madh3^(tmIpar) adenocarcinoma Laboratorities Cell 94 of large BarHarbor, (1988), 703 intestine ME Mouse FVB/N- spontaneous Jackson Zhanget al., TgN(UPII- carcinoma of Laboratorities Cancer Res. SV40T) bladderwith Bar Harbor, 59 (1999), 29Xrw metastasis to ME 3512 the liver MouseFVB- spontaneous Jackson Guy et al., NeuN adenocarcinoma LaboratoriesPNAS USA (N#202) of mammary Bar Harbor, 89 (1992), tissue ME 10578 RatRat F344/ spontaneous Charles River Hosokawa et CrCrlBR carcinoma ofLaboratorities al., pituitary Wilmington, Toxicol. MA Pathol. 21 (1993),283

EXAMPLE 7 Verification of Bacterium-Mediated Tumor Targeting andBacterium-Secreted Protein Therapy in Rodents With Implanted orSpontaneous Tumors

(A) Rationale

As shown in the previous examples, intravenous injection oflight-emitting bacteria results in entry, replication, and accumulationonly in the tumor regions in animals. This process can be monitored byimaging of light emission in tumors. Placing the endostatin and chimerictoxin expressing gene cassettes in cis configuration with alight-emitting gene cassette provides an indirect detection system invivo for their temporal and spatial delivery via bacteria.

The endostatin and chimeric toxin gene cassettes are linked to signalpeptide encoding sequences, which facilitate the secretion of theseproteins into the extracellular space in the tumor or into the cytosolof infected tumor cells. Both proteins secreted from bacteria into theextracellular space of the tumor are expected to function similarly todirectly injected purified proteins. Both proteins secreted from L.monocytogenes into the cytosol of the infected tumor cells will resemblethe viral delivery system reported earlier for endostatin. The bacterialsystems can be used as a constitutive secretion system or as anexogenously added IPTG-activatable secretion system in the tumor. Byregulating the expression levels of the therapeutic proteins in bacteriathat colonize the tumor, the secreted amount of proteins inhibitingtumor growth can be determined. Without the addition of IPTG, theinhibitory protein secretion from the intravenously injected bacteriawill be kept at minimum while in blood circulation. This will provide anadded safety to the recipient tumorous animals during delivery ofbacteria. Using the BSPT system, the onset and duration of the therapycan be controlled by the addition of IPTG. Upon completion of thetreatment, the bacterial delivery system can be eliminated byadministration of antibiotics, similar to treating a bacterialinfection.

(B) Determination of the Effect of Endostatin and PseudomonasExotoxin/TGF Alpha Fusion Protein Secreted by E. coli and L.monocytogenes on Tumor Growth in Animals with Implanted Tumors

The inhibitory effect of endostatin and the cytotoxicity of the chimerictoxin secreted by E. coli and L. monocytogenes in tumors will bedetermined as follows. Thirty-five nude mice bearing 10-day-old C6tumors will be injected with bacterial constructs as follows: (a) Fivemice with E. coli engineered to secrete endostatin; (b) Five mice withE. coli engineered to secrete chimeric toxin; (c) Five mice with L.monocytogenes engineered to secrete endostatin; (d) Five mice with L.monocytogenes-engineered to secrete chimeric toxin; (e) Five mice withE. coli secreting endostatin and chimeric toxin; (f) control group: fivemice injected with E. coli expressing bacterial luciferase alone, andfive mice with L. monocytogenes expressing GFP. At the time of bacteriainjection, each tumor volume will be determined. Three days afterinjection, the replication of bacteria in the tumors will be monitoredunder a low light imager or under a fluorescence stereomicroscope. Thelight emission and the tumor volume will be measured daily up to 20 daysafter bacterial injection. Ten days after injection, one animal fromeach group will be sacrificed and the levels of the secreted proteinspresent in the tumor tissue will be analyzed using Western blotanalysis. These experiments will result in inhibition of tumor growth inendostatin treated animals or a more dramatic tumor regression inanimals treated with chimeric toxin proteins. The tumor growth incontrol animals is not expected to be affected by the bacteria alone.

In a follow-up experiment, mice with spontaneous adenocarcinoma ofmammary tissue (strain FVB-neuN(N#202), Table 1) will be used to studythe effect of secreted proteins on tumor growth. An experimental schemeidentical to that described for the C6 tumor analysis will be used. Atthe completion of tumor therapy, the presence of endostatin or chimerictoxin in the tumor tissue will be determined by Western blot analysis.An identical experimental design will be used to assay the effect ofIPTG-induction of endostatin and chimeric toxin production in bacteriain C6 tumors as well as in the spontaneously occurring breast tumormouse model. It is expected that multiple IPTG induction of proteinexpression in bacteria might be required for successful tumor therapy.

At any stage of tumor treatment, it may be required to remove the lightemitting and therapeutic gene containing bacteria from the animal. Tocarry out this experiment, mice with 12-day-old C6 tumors will beintravenously injected with E. coli expressing the bacterial luciferase.Three days after injection, antibiotic therapy will be initiated byintraperitoneal administration of gentamicin (5 mg/kg body weight) twicedaily, or the newly discovered clinafloxacin (CL960) (Nichterlein etal., Zentralbl. Bakteriol. 286 (1997), 401). This treatment will beperformed for 5 days, and the effect of antibiotics on the bacteria willbe monitored by imaging light emission from the animals daily.

By completing the above experiments, it is expected that endostatin andchimeric toxin proteins secreted into the tumors will cause theinhibition of tumor growth and measurable tumor regression. It isanticipated that tumor regression will be achieved in both groups ofrodents with implanted tumors and with spontaneously occurring tumors.Experiments with simultaneous application of secreted endostatin andchimeric toxin proteins in tumor treatment may give the most promisingresults. The removal of the engineered bacteria from the tumor byadministration of antibiotics is an added safety measure of thebacterium-secreted protein therapy (BPST) of the present invention.

That which is claimed is:
 1. A method for treating a tumor, tumortissue, cancer or metastasis, comprising: administering a vaccinia virusintravenously to a subject comprising a tumor, tumor tissue, cancer ormetastatasis, whereby the tumor, tumor tissue, cancer or metastasis istreated, whereby there is regression of the tumor, cancer, ormetastases, wherein: the virus is a Lister strain variant from theInstitute of Viral Preparations (LIVP); and the virus replicates in thesubject and accumulates in the tumor, tumor tissue or matastases.
 2. Themethod of claim 1, wherein the virus encodes a protein for therapy of atumor, tumor tissue, cancer or metastasis.
 3. The method of claim 2,wherein the protein for therapy is selected from among a cytotoxicprotein, a cytostatic protein, an inhibitor of angiogenesis, a proteinthat stimulates apoptosis, a protein that inhibits an elongation factor,a protein that binds to a ribosomal subunit, a nucleotide-modifyingprotein, a nuclease, a protease, a cytokine, a toxin, an enzyme or areceptor.
 4. The method of claim 2, wherein the protein for therapy of atumor is Pseudomonas exotoxin, endostatin, interleukin 2, interleukin 12or glucuronidase.
 5. The method of claim 1, wherein the virus allows forvisualization of a tumor, tumor tissue, cancer or metastasis.
 6. Themethod of claim 5, wherein the virus furthermore encodes a protein fortherapy of a tumor, tumor tissue, cancer or metastasis.
 7. The method ofclaim 1, wherein the virus allows for external visualization of a tumor,tumor tissue, cancer or metastasis.
 8. The method of claim 1, whereinthe virus allows for detection of a tumor, tumor tissue, cancer ormetastasis based on a signal.
 9. The method of claim 8, wherein thesignal is detectable by magnetic resonance imaging (MRI).
 10. The methodof claim 1, wherein the virus allows for detection of a tumor, tumortissue, cancer or metastasis through detection of light.
 11. The methodof claim 1, wherein the virus encodes a detectable protein or a proteinthat induces a detectable signal.
 12. The method of claim 11, whereinthe virus encodes a protein that binds to a contrasting agent,chromophore or a compound or ligand for visualization of tissues. 13.The method of claim 11, wherein the virus encodes a protein that emitsor induces emission of light.
 14. The method of claim 11, wherein thevirus encodes a fluorescent or luminescent protein.
 15. The method ofclaim 11, wherein the virus encodes a luciferase.
 16. The method ofclaim 11, wherein the virus encodes a red fluorescent protein or a greenfluorescent protein.
 17. The method of claim 11, wherein the virusencodes a metal-binding protein.
 18. The method of claim 1, wherein thetumor is a breast tumor, prostate tumor, bladder tumor, glioma tumor,ovarian tumor, pancreatic tumor, an adenocarcinoma, liver tumor, colontumor or skin tumor.